Amino acid signaling in Saccharomyces cerevisiae: a permease-like sensor of external amino acids and F-Box protein Grr1p are required for transcriptional induction of the AGP1 gene, which encodes a broad-specificity amino acid permease.

The SSY1 gene of Saccharomyces cerevisiae encodes a member of a large family of amino acid permeases. Compared to the 17 other proteins of this family, however, Ssy1p displays unusual structural features reminiscent of those distinguishing the Snf3p and Rgt2p glucose sensors from the other proteins of the sugar transporter family. We show here that SSY1 is required for transcriptional induction, in response to multiple amino acids, of the AGP1 gene encoding a low-affinity, broad-specificity amino acid permease. Total noninduction of the AGP1 gene in the ssy1Delta mutant is not due to impaired incorporation of inducing amino acids. Conversely, AGP1 is strongly induced by tryptophan in a mutant strain largely deficient in tryptophan uptake, but it remains unexpressed in a mutant that accumulates high levels of tryptophan endogenously. Induction of AGP1 requires Uga35p(Dal81p/DurLp), a transcription factor of the Cys6-Zn2 family previously shown to participate in several nitrogen induction pathways. Induction of AGP1 by amino acids also requires Grr1p, the F-box protein of the SCFGrr1 ubiquitin-protein ligase complex also required for transduction of the glucose signal generated by the Snf3p and Rgt2p glucose sensors. Systematic analysis of amino acid permease genes showed that Ssy1p is involved in transcriptional induction of at least five genes in addition to AGP1. Our results show that the amino acid permease homologue Ssy1p is a sensor of external amino acids, coupling availability of amino acids to transcriptional events. The essential role of Grr1p in this amino acid signaling pathway lends further support to the hypothesis that this protein participates in integrating nutrient availability with the cell cycle.

The molecular genetics of hexose transport in yeasts.

Transport across the plasma membrane is the first, obligatory step of hexose utilization. In yeast cells the uptake of hexoses is mediated by a large family of related transporter proteins. In baker's yeast Saccharomyces cerevisiae the genes of 20 different hexose transporter-related proteins have been identified. Six of these transmembrane proteins mediate the metabolically relevant uptake of glucose, fructose and mannose for growth, two others catalyze the transport of only small amounts of these sugars, one protein is a galactose transporter but also able to transport glucose, two transporters act as glucose sensors, two others are involved in the pleiotropic drug resistance process, and the functions of the remaining hexose transporter-related proteins are not yet known. The catabolic hexose transporters exhibit different affinities for their substrates, and expression of their corresponding genes is controlled by the glucose sensors according to the availability of carbon sources. In contrast, milk yeast Kluyveromyces lactis contains only a few different hexose transporters. Genes of other monosaccharide transporter-related proteins have been found in fission yeast Schizosaccharomyces pombe and in the xylose-fermenting yeast Pichia stipitis. However, the molecular genetics of hexose transport in many other yeasts remains to be established. The further characterization of this multigene family of hexose transporters should help to elucidate the role of transport in yeast sugar metabolism.

Glucose-dependent and -independent signalling functions of the yeast glucose sensor Snf3.

The yeast Snf3 protein has been described to function as a sensor for low concentrations of extracellular glucose. We have found that Snf3 is able to transduce a signal in the complete absence of extracellular glucose. High basal activity of the HXT7 promoter during growth on ethanol required Snf3 as well as other components of the signalling pathway activated by Snf3. Moreover, the C-terminal domain of Snf3 was sufficient to complement the role of Snf3 in this regulation. As the C-terminal tail of Snf3 interacted with other components at the plasma membrane independent of the carbon source, our data suggest that Snf3 is involved in signalling complexes which can be activated by other signals than extracellular glucose.

Catabolite inactivation of the high-affinity hexose transporters Hxt6 and Hxt7 of Saccharomyces cerevisiae occurs in the vacuole after internalization by endocytosis.

After addition of high concentrations of glucose, rates of high-affinity glucose uptake in Saccharomyces cerevisiae decrease rapidly. We found that the high-affinity hexose transporters Hxt6 and Hxt7 are subject to glucose-induced proteolytic degradation (catabolite inactivation). Degradation occurs in the vacuole, as Hxt6/7 were stabilized in proteinase A-deficient mutant cells. Degradation was independent of the proteasome. The half-life of Hxt6 and Hxt7 strongly increased in end4, ren1 and act1 mutant strains, indicating that the proteins are delivered to the vacuole by endocytosis. Moreover, both proteins were also stabilized in mutants defective in ubiquitination. However, the initial signal that triggers catabolite inactivation is not relayed via the glucose sensors Snf3 and Rgt2.

Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae.

The hexose transporter family of Saccharomyces cerevisiae comprises 18 proteins (Hxt1-17, Gal2). Here, we demonstrate that all these proteins, except Hxt12, and additionally three members of the maltose transporter family (Agt1, Ydl247, Yjr160) are able to transport hexoses. In a yeast strain deleted for HXT1-17, GAL2, AGT1, YDL247w and YJR160c, glucose consumption and transport activity were completely abolished. However, as additional deletion of the glucose sensor gene SNF3 partially restored growth on hexoses, our data indicate the existence of even more proteins able to transport hexoses in yeast.

Glycogen metabolism in a Saccharomyces cerevisiae phosphoglucose isomerase (pgil) disruption mutant.

Disruption of the gene pgil of Saccharomyces cerevisiae, which codes for phosphoglucose isomerase, results in a dramatic increase in the amount of intracellular glycogen in early exponential cultures. The level of glucose 6-phosphate was much higher in mutant than in wild-type cells. Phosphorylase a activity and the state of activation of glycogen synthase were also investigated. Phosphorylase a activity was rather low along the culture in wild-type cells, whereas it was consistently higher in mutants. Glycogen synthase was mostly in the active form in early-medium exponential cultures in wild-type cells whereas the activation state of this enzyme in mutant cells, although lower at the earlier steps of the culture, did not differ from wild-type cells at later stages. The fact that the intracellular levels of UDP-glucose are markedly increased in mutant cells suggest that the observed accumulation of glycogen results from a rise in substrate availability rather than from the activation of the enzyme responsible for the synthesis of the polysaccharide.

Thoraco-omphalopagus conjoined twins: successful surgical separation.

Male thoraco-omphalopagus conjoined twins were evaluated systematically with respect to visceral joining. Cardiac studies included electrocardiography, echocardiography, and a dynamic radioactive heart scan. No union was found. Liver union was demonstrated by scanning and angiographic studies. No communication between the gastrointestinal tracts was found, and no other major anomalies were discovered. When the twins were approximately 3 months of age, surgical separation was performed. A common pericardium, two separate hearts, and a union of the liver were found. Prosthetic sheeting was used to close the pericardium and the thoracic-abdominal defects in both. In one, primary skin closure was possible. In the other silicone rubber sheeting was used to close a large skin defect in the chest. Both have done well over a follow-up period of 14 months. One is larger than the other, but the growth rate of both has improved in the last 6 months.

The effectiveness of Nissen fundoplication in neurologically impaired children with gastroesophageal reflux.

Fifty-seven of 101 Nissen fundoplications during the 4-year period, July 1979 to July 1983, were performed on neurologically impaired children. Mean age at the time of surgery was 5.9 years (range 1 month to 22 years). Indications for operation included: persistent vomiting, 57 patients (100%); failure to thrive, 49 patients (86%); repeated episodes of pneumonia, 49 patients (86%); esophagitis, 18 patients (32%); hiatal hernia, 14 patients (25%); episodes of apnea, 10 patients (18%); and esophageal stricture, six patients (10%). Forty-six of the 57 patients had previously failed a standard trial of nonsurgical management. Gastroesophageal reflux was documented by barium esophagograms in 51/56 patients (91%), chalasia scans in 28/32 patients (88%), esophagitis or stricture at endoscopy in 21/23 patients (91%), and acid reflux on pH monitoring in 13/16 patients (80%). Operative management included gastrostomy in 55 of the 57 patients and this was permanent in 50. Gastrostomies had previously been performed in nine patients but had failed to provide a reliable method of enteral feeding because of chronic reflux and aspiration. The surgical complication rate was 12%. Intraoperative esophageal perforation occurred in two patients, splenic tear in one, hepatic vein laceration in one, and a tight wrap in one. After surgery, bowel obstruction from adhesions developed in one patient and a midgut volvulus in another. Five of the children have died, none from causes related to the surgical procedure. Clinical and radiologic follow-up evaluations of all survivors have been done, with a mean follow-up of 3 years. In four patients the repair was felt to be inadequate. One patient had an esophageal stricture and three had recurring episodes of pneumonia. Three children showed radiologic evidence of persistent reflux, but only two were symptomatic. Two patients required a second antireflux procedure for reflux and are now free of symptoms. Nissen fundoplication appears to be a safe and beneficial procedure in neurological impaired children. Long-term follow-up evaluation of these patients showed satisfactory growth as well as a significant decrease in pulmonary disease associated with aspiration.

Selective management of injured spleen.

During the past 6 years, 68 consecutive children suffering from splenic trauma have been treated according to a selective management plan. Twenty-two patients (32%) underwent splenectomy, parenchymal repair was performed on 16 occasions (24%), and nonoperative treatment was employed in 30 children (44%). Overall results have been good in all three groups. Two children (3%) died as a result of their injuries, and 10 complications were recorded. Both deaths and 7 of the 10 complications occurred in the patients undergoing splenectomy. By utilization of a basic management plan that favors nonoperative treatment over splenic repair and repair over splenectomy, splenic salvage has been successfully accomplished in 86% of the 49 patients treated since 1977.

Dumbbell neuroblastomas in children.

A "dumbbell" neuroblastoma is a malignant neoplasm usually primary in the mediastinum or retroperitoneum with posterior extension through an intervertebral foramen to additional tumor within the spinal canal. Neurologic deficits are found in almost all cases. Nineteen patients with such tumors were reviewed with respect to diagnosis, management, and results. Treatment consisted of prompt laminectomy with total or subtotal excision of the extradural tumor. The primary tumor in the mediastinum or retroperitoneal area was removed at a second stage. All patients received postoperative radiation therapy. Eleven of the 17 patients observed more than two years are free of disease, a cure rate much higher than the overall experience with neuroblastoma. Substantial recovery of neurologic function occurred in most, but kyphoscoliotic deformities were a disturbingly frequent late complication.

Extrahepatic portal hypertension in children. Long-term evaluation.

A long-term evaluation of 43 children with extrahepatic portal hypertension indicates a high success rate and excellent subsequent health when a splenorenal or mesocaval shunt can be performed. On the other hand, makeshift shunts inevitably fail. Direct operations are considerably less successful than standard shunts. Esophagogastric resections with interpositions result in long-term freedom from bleeding in only about half the cases, and portoazygous disconnection procedures have been uniformly disappointing. Sclerotherapy in a relatively recent experience has been quite successful, but long-term results are presently unavailable. The condition carries a significant mortality rate. Complications from failed operations, division of the vena cava, and multiple transfusions are numerous. The general health of long-term survivors is excellent in those with successful operations, and is surprisingly good for patients whose operations have been unsuccessful and for those who have had no operations.

Acetylthiocholinesterase staining activity of rectal mucosa. Its use in the diagnosis of Hirschsprung's disease.

Increased acetylthiocholinesterase (AchE) reactivity in the rectal lamina propria and lamina muscularis mucosae was used to diagnose Hirschsprung's disease. We processed 131 specimens with the AchE reaction; 43 were suction biopsy specimens and the rest were full-thickness specimens. Of the 68 specimens in which neurocytes were present, none demonstrated a diffuse increase in the number of nerve fibers. However, 15 showed focal increases in the numbers of fibers that were not large enough to be regarded as indicative of Hirschsprung's disease. All patients with a diffuse increase in nerve fibers, regardless of the type of biopsy, were shown to have Hirschsprung's disease. The AchE staining reaction did, however, produce a 29% rate of false-negative reactions (16 of 56 specimens) in patients with Hirschsprung's disease. These data demonstrate that an abnormal pattern of AchE reaction is diagnostic of aganglionic megacolon, whereas a normal pattern does not exclude the disease.

Glucagon in experimental intussusception.

A standard type of ileocolic intussusception was produced in 69 puppies and the effect of glucagon on the hydrostatic reduction of the intussusceptions evaluated in a prospective, double-blind study. The reductions were attempted at intervals varying from 18 to 60 hr following the production of the intussusceptions. The overall reduction rate was 70% and there was no statistical difference in this rate between the animals receiving glucagon and those receiving placebos. None of the gangrenous intussusceptions were reducible with the hydrostatic pressure technique, irrespective of whether glucagon was used or not. A further evaluation was made of those animals in whom successful reductions were accomplished by the hydrostatic pressure method. Glucagon did result in significantly easier reductions, and an earlier return of normal vascular supply as measured by color and by arterial pulsations as compared to the control group.

Iatrogenic ileal atresia secondary to clamping of an occult omphalocele.

Clamping of an occult omphalocele has led to complete division of an entrapped loop of ileum in two instances. The proximal end has been sealed in the process, producing an iatrogenic ileal atresia. The clinical picture in both instances differed from that usually found with ileal atresia. Escape of a small amount of meconium from the transected cord was noted in one, and the stump of the cord in the other appeared red and engorged. Clamping the umbilical cord routinely at least 5 cm from the abdominal wall is recommended.

Congenital vas deferens--ureteral connection.

Congenital drainage of the vas deferens into a ureter permits retrograde passage of urine through the vas to the epididymis. If this anomaly is accompanied by urinary tract pathology with vesicoureteral reflux, hydroureter, stasis, and infection, the passage of urine into the vas can very well lead to epididymitis of a chronic or recurring nature and secondary scrotal abscesses. Two cases of this rare anomalous connection are presented in which such scrotal infections occurred. The diagnosis should be suspected on clinical grounds and can be confirmed by cystograms in which the dye refluxes up the ureter and passes into the vas. The association of rectal anomalies is of embryologic significance and may be additionally important clinically by further suggesting the possibility of the ureteral-vas anomaly.

Secondary thyroid neoplasms in pediatric cancer patients: increased risk with improved survival.

Between 1973 and 1983, eight children who had undergone successful multimodal management of malignant tumors developed secondary thyroid neoplasms. The primary tumors were acute lymphocytic leukemia in three, Wilms' tumor in two, and Hodgkin's disease, rhabdomyosarcoma, and ganglioneuroblastoma in one each. During this period, 174 long-term survivors with these five diagnoses were enrolled in our tumor registry, yielding a 4.6% incidence of secondary thyroid neoplasms. All eight patients received both radiation and chemotherapy. The mean radiation dose was 2,700 r with a calculated thyroid dose of 2,140 r (range, 5 to 4,200 r). Age of diagnosis of the primary tumors ranged from 1 to 8 2/12 years (mean, 5 years), and the latent period between treatment and development of the thyroid lesions averaged 6 1/2 years. Thyroid neoplasms presented at an average age of 11 4/12 years. Five patients developed solitary adenomas, one presented with multiple adenomas, and two had follicular carcinoma with regional lymph node metastases. Although thyroid neoplasms are rare in childhood, clinically apparent thyroid tumors have been observed in up to 2.5% of children following radiation exposure (mean follow-up, 24 years). The reported latent period before the development of thyroid neoplasms in irradiated patients is at least 10 years, with the peak incidence occurring 20 to 25 years after exposure. This study documents a 4.6% incidence of subsequent thyroid neoplasms in pediatric cancer patients within a relatively short follow-up period (mean, 11 years). These thyroid tumors occurred at an earlier age (mean, 11.5 years) and with a shorter latent period (mean, 6.5 years) than would be predicted from previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Gastroschisis update.

Sixty-four infants with gastroschisis have been managed in the 9-yr period, 1970-1979, with four postoperative deaths. The silo technique has been the standard method of management in this series, permitting initial expansion of the abdominal cavity without increased abdominal pressure and respiratory embarassment. Removal of the silo and complete closure of the abdominal wall deficit were possible 5-12 days later. A high proportion of the infants were below 2500 g in weight at birth (61%); and although 3 of the 4 postoperative deaths occurred in the low birth weight group, this did not appear to be a factor in mortality. Associated anomalies occurred in 25 of the 64, but only the coincident intestinal atresias (6) were of major significance. These additional anomalies were not responsible for deaths. Two deaths occurred from problems dating from birth, one from aspiration and the second from sepsis. The other two resulted from postoperative complications resulting in infarction of the midgut. Both were caused in part by failure to adequately enlarge the abdominal wall defect at the time of the initial procedure. Other postoperative complications were relatively few. Although all required intravenous nutritional support, the long term results in terms of growth and development and of intestinal function were quite satisfactory.

Atresia of the colon.

Eleven infants with colon atresia have been managed by staged procedure with survival and good health in ten. Complications have been relatively few. At the initial operation the proximal atretic segment is exteriorized as an end colostomy. Such a procedure is simple and safe, results in rapid relief of the obstruction, and permits normal feeding by mouth within a few days. At a second procedure several weeks or months later, intestinal continuity is established by an end-to-end or end-to-side anastomosis. Gastroschisis has been an associated anomaly in four of these cases. This association gives additional support to the concept that intrauterine interference to the blood supply to a segment of intestines is the etiologic factor responsible for such atresias.

The use of glucagon in the diagnosis and management of illeocolic intussusception.

Twenty-one of 25 episodes of ileocolic intussusception were successfully reduced by the barium enema hydrostatic technique supplemented by glucagon. No apparent serious complications occurred from the use of this drug. The comfort of the children and the ease of filling the colon and small bowel seemed enhanced by the glucagon.

Pancreatic pseudocyst complicating treatment of acute lymphoblastic leukemia.

In the management of children with acute lymphoblastic leukemia, L-asparaginase has become established as an effective drug in the usual multi-agent therapy; and the significance of pancreatitis as a complication of this drug is well recognized. Less well appreciated, however, is the progression of such pancreatitis in some patients to pseudocyst formation and the possible necessity for surgical management. Two adolescent girls who developed pancreatic pseudocysts while being treated with L-asparaginase are described in this report. Both were being treated for acute lymphoblastic leukemia for periods of 18 and 4 months, respectively, prior to the onset of pancreatitis. Both were in remission of their leukemic disease when typical clinical and laboratory manifestations of acute pancreatitis developed. In one girl, a pancreatic pseudocyst became apparent 2 weeks following the diagnosis of acute pancreatitis and in the other girl, this complication developed over a period of 8 weeks. The usual nonsurgical management of pancreatitis over protracted periods of time was ineffective in the treatment of the pseudocysts. Surgical drainage (internal in one and external in the other) was successful in both in eradicating the pseudocyst, and in neither did further evidence of pancreatic disease subsequently occur. In both resumption of chemotherapy, omitting L-asparaginase, was well tolerated. One has been in remission of leukemia and in good health for a 3-year period of follow-up observation, while the other subsequently had a relapse of leukemia and died 18 months following the onset of pancreatitis.